The main objectives of this continuation application are to elucidate the molecular mechanisms by which TSA enhances apoptosis induced by the proteasome inhibitor PS-341 in head and neck squamous cell carcinoma (HNSCC) cells. During the last funding period, we found that the co-treatment of PS-341 and TSA in HNSCC cells synergistically induces apoptosis by increasing the expression of pro-apoptotic gene Noxa. To further understand the molecular mechanisms by which TSA enhances PS-341-induced apoptosis, we propose the following four specific aims: Aim 1 is to explore the epigenetic mechanisms by which PS- 341/TSA induce Noxa expression in HNSCC cells. We hypothesize PS-341/TSA may modify histone ubiquitination to promote Noxa expression in addition to histone accetylation. We will perform chromatin immunoprecipitation (ChIP) assay to determine how PS-341 modulates ubiquitination of H2A and promotes Noxa expression. Aim 2 is to determine whether TSA may enhance apoptosis by inhibiting autophagy formation in HNSCC cells. Based on our preliminary studies, we hypothesize that TSA may inhibit autophagy and heat shock responses by targeting cellular HDAC6. We will examine whether TSA reduces PS-341- induced autophagy, thereby enhancing apoptosis. We will examine whether TSA inhibits heat shock protein expression by targeting the transcription factor heat shock factor 1. Aim 3 is to determine whether PS- 341/TSA potently induce the apoptosis of cancer initiating cells (CICs) in vitro and in vivo. CICs exhibit an intrinsic resistance to chemotherapeutic agents, preventing complete elimination of the tumor. Paradoxically, CICs are difficult to maintain or propagate in vitro which represents a significant barrier to study CICs properties and to screen cancer therapeutic drugs against CICs. To overcome this barrier, we propose to develop a cell model system to study CICs from human HNSCC cell lines. We will examine whether PS- 341/TSA can potently induce apoptosis of CICs in vitro and in vivo. Aim 4 is to explore whether and how Hippo-YAP signaling modulates PS-341/TSA-induced apoptosis. We will over-express or knock-down YAP in HNSCC cells to determine how YAP modulates PS-341/TSA-induced apoptosis. Our work may help to develop targeting therapy for HNSCC with abnormal activation of YAP signaling. In summary, new findings from this application will have clinical implications for treating HNSCC and may help to develop innovative strategies for human cancer treatment RELEVANCE (See instructions): Head and neck cancer typically presents as a very malignant tumor and is frequently resistant to chemo- therapy. The major goals of this project are to develop new therapy strategies for treating head and neck cancer and to improve efficacy of chemotherapy.